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Biomedical subjects

J Hartman

Publications and source records attributed to J Hartman.

At least 19 recordsLinked to original sources
PubMed1994

Cystic fibrosis transmembrane conductance regulator mutations that disrupt nucleotide binding.

Increasing evidence suggests heterogeneity in the molecular pathogenesis of cystic fibrosis (CF). Mutations such as deletion of phenylalanine at position 508 (delta F508) within the cystic fibrosis transmembrane conductance regulator (CFTR), for example, appear to cause disease by abrogating normal biosynthetic processing, a mechanism which results in retention and degradation of the mutant protein within the endoplasmic reticulum. Other mutations, such as the relatively common glycine-->aspartic acid replacement at CFTR position 551 (G551D) appear to be normally processed, and therefore must cause disease through some other mechanism. Because delta F508 and G551D both occur within a predicted nucleotide binding domain (NBD) of the CFTR, we tested the influence of these mutations on nucleotide binding by the protein. We found that G551D and the corresponding mutation in the CFTR second nucleotide binding domain, G1349D, led to decreased nucleotide binding by CFTR NBDs, while the delta F508 mutation did not alter nucleotide binding. These results implicate defective ATP binding as contributing to the pathogenic mechanism of a relatively common mutation leading to CF, and suggest that structural integrity of a highly conserved region present in over 30 prokaryotic and eukaryotic nucleotide binding domains may be critical for normal nucleotide binding.

Adenosine Triphosphate↗
PubMed1992

Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product.

The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product. We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508). Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP. Irreversible NBD-1 labeling by an ATP analog was demonstrated using [32P]8-azido-ATP. This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain. These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide. Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding.

Adenosine Triphosphate↗
PubMed1992

Intrinsic anion channel activity of the recombinant first nucleotide binding fold domain of the cystic fibrosis transmembrane regulator protein.

The first nucleotide binding fold (NBF-1) from the cystic fibrosis transmembrane regulator (CFTR) has been expressed in bacteria and found to bind ATP and to express anion channel activity when reconstituted onto a planar lipid bilayer. This evidence suggests that the NBF forms the anion-selective portion of the CFTR channel. We also found that the recombinant NBF-1 anion channel is blocked by ATP (1 mM), under which condition it appears to have a minimal conductance of approximately 9 pS and an ohmic current-voltage relationship. We further found that the recombinant NBF-1 bearing the delta F508 mutation has nearly identical anion channel activity to that of the wild-type protein but can be distinguished from wild type under bianionic conditions with chloride and gluconate. We conclude from these data that the anion channel activity of the recombinant NBF-1 could represent all or part of the anion conductance mechanism of CFTR and that the role of the ATP binding by the NBF could be to modulate this anion channel activity.

Adenosine Triphosphate↗
PubMed1992

Hypothyroidism in adults with sickle cell anemia.

Persons with sickle cell anemia have several indications for transfusion of red blood cells. One of the complications of transfusion of red blood cells is iron overload. Iron overload has been associated with multiple endocrine abnormalities. We report herein three cases of hypothyroidism in adult individuals with sickle cell disease. All three patients were over the age of 45 years at the time of the diagnosis and had received multiple units of transfused red blood cells and had serum ferritin levels of greater than 6,000 ng/mL. All patients were diagnosed during times when they were critically ill. Replacement therapy was instituted in all cases; however, all three patients died shortly after the diagnosis of hypothyroidism was made. Congestive heart failure appeared to be a primary cause of death in all three patients. In the one patient in whom a postmortem examination was done, there was evident extensive fibrosis of the thyroid gland as well as extensive deposition of iron in the cells lining the thyroid follicles. We believe that this represents the first report of clinical hypothyroidism in patients with sickle cell anemia who have received multiple transfusions. Awareness of this condition is especially important given that congestive heart failure is common in sickle cell disease.

Anemia, Sickle Cell↗
PubMed1992

Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex.

Nitric oxide (NO) is an important messenger both systemically and in the CNS. In digital Ca2+ imaging and patch-clamp experiments, clinically available nitroso compounds that generate NO are shown to inhibit responses mediated by the NMDA subtype of the glutamate receptor on rat cortical neurons in vitro. A mechanism of action for this effect was investigated by using the specific NO-generating agent S-nitrosocysteine. We propose that free sulfhydryl groups on the NMDA receptor-channel complex react to form one or more S-nitrosothiols in the presence of NO. If vicinal thiol groups react in this manner, they can form a disulfide bond(s), which is thought to constitute the redox modulatory site of the receptor, resulting in a relatively persistent blockade of NMDA responses. These reactions with NO can afford protection from NMDA receptor-mediated neurotoxicity. Our results demonstrate a new pathway for NO regulation of physiological function that is not via cGMP, but instead involves reactions with membrane-bound thiol groups on the NMDA receptor-channel complex.

Alkylating Agents↗
PubMed1990

Regulation of tissue plasminogen activator in sickle cell anemia.

Evidence of activation of the clotting system in individuals with sickle cell anemia (SCA) has been observed by several investigators. It has been suggested that the clotting and fibrinolytic systems may play a role in the pathophysiology of vaso-occlusion in SCA. We reported previously evidence of abnormal fibrinolytic activity as reflected in decreased releasable tissue plasminogen activator (t-PA) using a functional assay. We have examined the mechanism of the decreased functional releasable t-PA in individuals with SCA. We studied 12 patients with respect to releasable t-PA, fast acting inhibitor to t-PA (or PAI-1), and immunoreactive or antigenic t-PA. These SCA individuals were at their baseline states and not taking medications known to interfere with the fibrinolytic or clotting systems. We found that the mean releasable t-PA for the SCA individuals was 0.01 IU/ml of plasma with a standard error of mean (SEM) of 0.01. The mean releasable t-PA of 118 healthy normal controls was 0.70 IU/ml with SEM 0.10 (P less than .001). The mean level of fast-acting inhibitor to t-PA in unoccluded circulation of the SCA patients' plasma was 16.5 IU/ml with SEM of 3.54. The mean plasma levels of fast-acting inhibitor to t-PA in 56 healthy controls was 2.56 IU/ml with SEM of 0.29 (P less than .0001). The SCA patients had a mean baseline t-PA antigen level of 5.98 ng/ml with SEM of 1.72. The mean level of t-PA antigen of 78 healthy controls using the same technique was 4.3 ng/ml with SEM of 2.7 (not significant). The mean baseline functional t-PA for SCA individuals was 0.15 IU/ml with SEM 0.01 and the mean baseline functional t-PA for 118 controls was 0.17 IU/ml with SEM 0.10. These data suggest that the mechanism of decreased releasable t-PA in sickle cell anemia is related to an elevation of fast-acting inhibitor to t-PA and that antigenically t-PA is present in normal quantities in the baseline plasma in this population.

Adult↗
PubMed1989

Force deficit of vascularized skeletal muscle grafts in rabbits.

Through autografting experiments on 9-g rectus femoris (RFM) muscles in rabbits, we substantiated a previous observation that the maximum isometric tetanic force (Po) and specific Po (N/cm2) of neurovascular-intact grafts are not different from grafts made with neurovascular repair. We then tested the hypotheses that the specific Po of vascularized grafts is significantly less than that of control RFM muscles and the deficit in the specific Po is associated with increases in connective tissue and interstitial space. The specific Po of the grafts was 65% of the value for control RFM muscles. Connective tissue protein concentration of grafts was 3.8 times greater than the control value of 16.6 +/- 3 micrograms/mg wet mass, but this only accounted for a 5% correction in specific Po. The volume of interstitial space did not differ between grafts and control muscles. We conclude that the deficit of 35% in specific Po of vascularized grafts compared with control values is partially explained by an increase in connective tissue, but a 30% unresolved deficit remains.

Animals↗
PubMed1988

Totally implantable intravenous catheters in the management of sickle cell anemia.

Totally implantable catheters (TICs) have recently been employed for long-term central venous access in patients with sickle cell disease (SCD). We have reviewed our experience with 10 TICs inserted in patients with SCD. These were compared to 33 TICs inserted in patients without SCD (controls). The primary diagnosis was malignancy in most of the controls. The SCD patients experienced a marked increase in total complications (70 vs. 24%), as well as in complications requiring catheter removal (50 vs. 3%). No variable explained these differences except the presence of SCD. The complications requiring catheter removal from SCD patients were infections, catheter thrombosis, and venous thrombosis. The increased risk of these complications must be considered before a catheter is inserted; however, the average useful life of these catheters exceeded 12 months. They remained useful in the care of patients with poor venous access and multiple complications of SCD.

Adult↗
PubMed1988

Failure to detect the presence of pluripotential haemopoietic stem cells in the mouse brain.

Single cell suspensions prepared from adult mouse brains were tested for the presence of pluripotential haemopoietic stem cells (colony-forming units, CFU) by transfer into an irradiated recipient and enumeration of the CFU in the recipient's spleen. In contrast to the findings of others (Bartlett, 1982), we did not detect CFU after injection of brain cell suspensions, although they were detectable after inoculation with bone marrow cells. The number of CFU in recipients after transfer of increasing numbers of brain cells was the same as that detected in the irradiated controls which had not received any transferred cells. Finally, cells from the brain, in contrast to bone marrow cells, were not able to protect recipient animals from the effects of lethal irradiation.

Animals↗
PubMed1987

Comparison of formal language therapy with supportive counseling for aphasia due to acute vascular accident.

A total of 60 right-handed patients with acute aphasia due to left hemispheral stroke were randomly assigned to two modes of therapy for six months, beginning one month after the ictus. Conventional speech therapy provided by professional speech pathologists twice weekly was compared with emotionally supportive counseling therapy, also provided by professional speech pathologists at the same intervals. Language function was tested periodically by the Porch Index of Communicative Ability. Fifty of the subjects were also tested at ten months after the ictus. There was no difference in the amount of improvement between the two groups.

Adult↗
PubMed1987

Correlation between changed biophysical properties of surface membranes and embryonic brain cell adhesivity. Influence of detergents, fixation, EGTA, colchicine, vinblastine, increased K+, ouabain and DMA on the primary cellular adhesivity of embryonic brain cell.

Embryonic mouse brain cells were rotated for 120 min and cellular adhesivity was tested under normal conditions and in the presence of substances which change the membrane properties. A marked decrease of cellular adhesivity (but not complete inhibition) was recorded in the presence of anionic detergents, while fixation of cells caused only non-significant inhibition Colchicine (1 mumol/l) and vinblastine (10 micrograms/ml) did not significantly affect the adhesivity. Increased external K+ (10 mumol/l) and ouabain (10 mmol/l) were also without a significant effect, however, EGTA (0.1 and 0.01 mmol/l) inhibited the adhesivity significantly. 2,3 dimethyl maleinic anhydride (DMA) which removes a part of the positive charge, caused a slight decrease of adhesivity. It is suggested that the primary adhesivity of brain cells is dependent upon the structural integrity of surface membranes, while the organization of the tubular system does not play a significant role. Isotonic concentration of monovalent cations is optimal for adhesivity and an increased concentration of external K+ or ouabain did not affect adhesivity significantly.

Animals↗
PubMed1987

Influence of increased concentration of Ca2+, La3+, PVP and urea on primary adhesivity of embryonic brain cells. Ultrastructural pattern of adhering cells.

With the help of a previously described experimental arrangement the influence of increased external concentration of Ca2+, La3+, PVP and urea was tested on the initial stages of brain cell adhesivity and its kinetics. Urea, an inhibitor of hydrogen bonds, significantly inhibited the adhesivity of the treated cells. PVP significantly increased cell adhesivity. The adhesivity was enhanced and speeded up by increased concentrations of Ca2+ and La3+. It is evident that the membrane surface potential, zeta potential and formation of H+ bonds and bridges are highly important for cellular adhesivity. EM control of freshly dissociated cells disclosed that a part of the cells had been damaged. According to the ultrastructural organisation, the surface membrane is damaged to a small or greater extent. Intercellular contacts were formed in vitro either between non damaged surfaces of membranes, or between fragments of membranes, or contacts were mediated by membrane debris. Because cellular debris disappeared during rotation from single adhesive complexes, it is probable, that disrupted membranes are used for restoration of membranes, or serve as a metabolic substrates, or are catabolized.

Animals↗
PubMed1986

Circahoralian changes in the size and dry mass of glioma C-6 cells in culture.

Changes in the size and dry mass of glioma C-6 intact cells in culture were investigated for 35-50 min at 5 min intervals by means of vital cytointerferometry. Rhythmic variations in the size, dry mass and protein concentration were thereby revealed in glioma cells. These variations fall into the category of circahoralian ones. While considerable variations in the cell area and dry mass were observed, changes in protein concentration were less pronounced. Addition of dibutyryl cylic AMP (db-cAMP), in a concentration of 10(-3) mol/l to the cultivation medium, produced no effect on the rhythm of the above parameters in glioma C-6 cells.

Animals↗
PubMed1986

Cell-to-cell adhesion of dissociated embryonic brain cells; the effects of metabolic inhibitors and temperature.

Brain cells from 16 to 18-day-old mice embryos were dissociated by mild trypsinization and rotated for 120 min. The area and density of of the adhesive complexes formed were registered using the method described previously. The adhesiveness of dissociated embryonic brain cells (measured during the 120 min of rotation) was diminished in the presence of inhibitors of protein synthesis (puromycin, cycloheximide and inhibition of mRNA synthesis actinomycin D). The inhibition was, however, not distinct, because 1 microgram/ml of cycloheximide and actinomycin was without any significant effect, and the degree of inhibition evoked by 10 micrograms/ml and 25 micrograms/ml of puromycin bordered on significance. However, protein synthesis inhibitors in long-term aggregation experiments had a pronounced inhibitory effect and/or induced destruction of the aggregates. Metabolic inhibitors (KCN and NaN3) caused an inhibition at the lowest level of significance (p less than 0.05) 10(-3) mol/l KCN reduced the final adhesive product significantly. Cells rotated at room temperature and at +5 degrees C adhere to the same extent as in control experiments (37 degrees C). The adhesion was significantly inhibited at +60 degrees C and also after freezing at -80 degrees C with subsequent thawing. The adhesion of cells exposed for 30 min to between +80 degrees C and 100 degrees C was completely abolished. The process of embryonic brain cell adhesion requires a low energy supply, and is relatively independent of biosynthetic processes and of temperature changes between +5 degrees C and +50 degrees C.

Animals↗